Introns are removed from eukaryotic mRNA precursors by a large, complex, and dynamic molecular machine known as the spliceosome. The fully assembled spliceosome consists of five small nuclear ribonucleoprotein particles (snRNPs) each containing a single small nuclear RNA (U1, U2, U4, U5, or U6 snRNA) together with a number of snRNP proteins. In collaboration with as many as 100 auxiliary proteins, the snRNPs assemble stepwise onto the mRNA precursor, and then participate in a changing network of interactions that catalyze the two chemical steps of the splicing reaction, release the spliced mRNA and excised intron, and prepare the five snRNPs for reuse. A major experimental problem in analyzing this complicated multistep process has been to identify reaction intermediates. Following the lead of Konarska, we have used oligonucleotides resembling splice sites, and antisense 2'-0-methyl oligonucleotides directed against certain snRNA sequences, to efficiently induce the assembly of spliceosome-like snRNP complexes in mammalian nuclear extracts. These artificially induced spliceosome-like complexes appear to freeze a variety of transient spliceosomal interactions (snRNP/snRNP, snRNP/substrate) that take place naturally over the course of an mRNA splicing reaction. Using this approach, we have identified two novel complexes: a U1/U4/U5 complex that appears to be an intermediate in displacement of U1 from the 5' splice site, and a U2/5' SS complex in which U2 bind the 5' exon in much the same way as the EBS2 element of a Group II intron binds the 5' exon sequence known as IBS2. We propose to characterize each of these complexes biochemically, to identify the sequence of events leading to formation of the complexes, and to develop assays to explore the role of these new RNA/RNA pairings in vivo. The experiments should shed new light on how the initial U4/U6 pairing comes undone early in spliceosome assembly; how U1 binding to the 5' splice site is replaced by U2, U5, and U6; how the active site is assembled from U2 and U6; and how the 5' exon is retained by U2 and U5 after the first chemical step of splicing, and aligned for ligation in the second step.